The effects of higher versus lower protein delivery in critically ill patients: an updated systematic review and meta-analysis of randomized controlled trials with trial sequential analysis

Background A recent large multicentre trial found no difference in clinical outcomes but identified a possibility of increased mortality rates in patients with acute kidney injury (AKI) receiving higher protein. These alarming findings highlighted the urgent need to conduct an updated systematic review and meta-analysis to inform clinical practice. Methods From personal files, citation searching, and three databases searched up to 29-5-2023, we included randomized controlled trials (RCTs) of adult critically ill patients that compared higher vs lower protein delivery with similar energy delivery between groups and reported clinical and/or patient-centred outcomes. We conducted random-effect meta-analyses and subsequently trial sequential analyses (TSA) to control for type-1 and type-2 errors. The main subgroup analysis investigated studies with and without combined early physical rehabilitation intervention. A subgroup analysis of AKI vs no/not known AKI was also conducted. Results Twenty-three RCTs (n = 3303) with protein delivery of 1.49 ± 0.48 vs 0.92 ± 0.30 g/kg/d were included. Higher protein delivery was not associated with overall mortality (risk ratio [RR]: 0.99, 95% confidence interval [CI] 0.88–1.11; I2 = 0%; 21 studies; low certainty) and other clinical outcomes. In 2 small studies, higher protein combined with early physical rehabilitation showed a trend towards improved self-reported quality-of-life physical function measurements at day-90 (standardized mean difference 0.40, 95% CI − 0.04 to 0.84; I2 = 30%). In the AKI subgroup, higher protein delivery significantly increased mortality (RR 1.42, 95% CI 1.11–1.82; I2 = 0%; 3 studies; confirmed by TSA with high certainty, and the number needed to harm is 7). Higher protein delivery also significantly increased serum urea (mean difference 2.31 mmol/L, 95% CI 1.64–2.97; I2 = 0%; 7 studies). Conclusion Higher, compared with lower protein delivery, does not appear to affect clinical outcomes in general critically ill patients but may increase mortality rates in patients with AKI. Further investigation of the combined early physical rehabilitation intervention in non-AKI patients is warranted. Prospero ID CRD42023441059. Supplementary Information The online version contains supplementary material available at 10.1186/s13054-023-04783-1.


Background
The role of protein dosage in critically ill patients is of considerable interest as it is thought to improve clinical outcomes by attenuating protein losses during critical illness and supporting the patients' recovery in later phases [1].Consequently, clinical nutrition societies generally recommend higher protein delivery, whereas these recommendations are based on a low level of evidence, leading to varying dosage recommendations (ranging from 1.2 to 2.5 g/kg body weight [BW]/day) and uncertainties in the clinical practice [2][3][4] due to the unclear benefits and risks [5].
A previous systematic review and meta-analysis (SRMA) included 19 randomized controlled trials (RCTs) and 1731 patients comparing higher (~ 1.3 g/kg BW/day) vs. lower (~ 0.9 g/kg BW/day) protein delivery (with similar energy delivery between groups) found that higher protein delivery was not associated with overall mortality but significantly attenuated muscle loss in five small RCTs [6].A trend towards shorter durations of mechanical ventilation (MV) and intensive care unit (ICU) length of stay (LOS) with higher protein delivery was also demonstrated [6].Following this SRMA, several RCTs were published, and one of them represents the large multinational, multicentre EFFORT protein trial.This trial compared higher (1.6 g/ kg BW/day) vs. lower (0.9 g/kg BW/ day) protein delivery and could not confirm any benefits or improved outcomes with higher protein delivery [7].Rather, these results indicate that higher protein delivery may increase mortality censored at 60 days in patients with acute kidney injury (AKI) and high organ failure scores [7].Although the EFFORT protein trial may itself already impact clinical practice, it is crucial to aggregate all available data to provide the best evidence to inform and guide clinical practice.Accordingly, the new relevant data from the EFFORT Protein trial and other recent RCTs need to be included in the updated SRMA to achieve greater precision on the pooled estimates.However, since the risks of type-I and -II errors may persist, trial sequential analysis (TSA) can be employed to detect such errors and thereby increase the certainty of the aggregated findings.Additionally, TSA quantifies the sample sizes required for clinically meaningful outcomes and offer insight into the potential futility of future trials, guiding feasibility, and choice of outcome measures.[8] Currently, evaluation of biochemical and patientcentred outcomes is lacking in published SRMAs.The lack of these outcomes precludes a comprehensive understanding of the associated biochemical sequelae of higher protein delivery.Similarly, the pooled estimate of combining early physical rehabilitation and higher protein delivery on patient-centred outcomes is lacking.Since early physical rehabilitation may improve protein utilization, it is essential to quantify their synergistic effects.
In light of these considerations, there is an urgent need to update the previous SRMA to address the following objectives: (1) compare the effect of higher vs. lower protein delivery (with similar energy between groups) on clinical outcomes in critically ill patients with and without acute kidney injury (AKI) and early physical rehabilitation and (2) summarize the biochemical sequelae and physical function outcomes of higher protein delivery.

Methodology
We conducted this SRMA according to the PRISMA 2020 guidelines [9].The PRISMA 2020 checklist is shown in Additional file 1: supplementary methods.The study protocol was registered in PROSPERO (CRD42023441059).

Eligibility criteria
We included RCTs of (1) adult (age ≥ 18) critically ill patients (mechanically ventilated or if uncertain, the control group mortality had to be greater than 5% to ensure including truly critically ill patients) that (2) compared protein doses with delivery via enteral (EN) formula, EN protein supplementation, parenteral nutrition (PN), or intravenous (IV) amino acids, (3) reported similar energy delivery between groups, and (4) reported clinical and/or patient-centred outcomes.
Studies among elective surgical or non-critically ill patients or studies with only laboratory, metabolic, or nutritional outcomes were excluded.Studies that investigated the effect of immunonutrition (e.g.glutamine or arginine) were also excluded.Quasi-randomized trials and studies published in abstract form were excluded.Post hoc, since our search also retrieved studies with a combination of protein and early physical rehabilitation, and the latter may enhance protein utilization, we also included studies with such combined interventions.

Information source and search strategies
An updated systematic search in MEDLINE, EMBASE, and CENTRAL through OVID was conducted with relevant subject headings and keywords from our last search (1 April 2022) [6] to (29 May 2023) without language restrictions.Personal files and the reference list of previous SRMAs were reviewed.Additional file 1: Table S1 shows the search strategies.ClinicalTrials.govwas also searched for ongoing studies (Additional file 1: Table S2).

Study selection process
Search results were exported into Covidence (Veritas Health Innovation, Melbourne, Australia) to remove duplicates and screen for potential eligible studies using the title and abstract of the articles (ZYL).The potential studies were retrieved, and two authors evaluated the full text independently (ZYL, ED).Disagreements were discussed with two other authors (CCHL and CS).

Data collection process
Data items were collected independently by two authors (ZYL, ED) in a standardized data abstraction form and thereafter summarized into tables.Details of data handling are in Additional file 1: Supplementary Methods.

Study quality and risk-of-bias assessment
The quality of the included trials was evaluated independently by two authors (ZYL, ED) using the Canadian Critical Care Nutrition (CCN) Methodological Quality System and the Cochrane Risk of Bias version 2 (ROB2).[10].The overall ROB2 assessment was categorized as low risk of bias, some concerns, or high risk of bias.The risk-of-bias traffic light and summary plots were generated by the Risk-of-bias VISualization (robvis) tool [11].The use of the CCN Methodological Quality System allows us to compare critical care nutrition trials across time and topics.The scoring table is shown in Additional file 1: Table S3.Any disagreements were discussed with two other authors (CCHL and CS).

Outcomes
Overall mortality is the primary outcome; all other outcomes are secondary.These latter outcomes are: (i) nutritional outcomes, (ii) clinical outcomes, (iii) muscle outcomes, (iv) discharge to rehabilitation facilities, (v) quality of life (QOL) physical measurements, and (vi) biochemical outcomes (details of each outcome are in Additional file 1: Supplementary Methods).Outcomes with at least 2 studies were pooled and reported.

Subgroup analysis
The following subgroup analyses were planned a priori: low vs other risk of bias, single vs multicentre trial, EN vs exclusive PN/intravenous amino acids, and AKI vs no/not known AKI.The subgroup analysis of AKI was performed in one study that enrolled exclusively AKI patients [12] and two studies that reported mortality outcomes in their subgroup of patients with AKI (Nephroprotect trial [12] and EFFORT protein trial [7]).For the Nephroprotect trial, we used the data from their secondary analysis that reported 90-day mortality outcome among patients with baseline kidney dysfunction (creatinine > 168 umol/L at the time of enrolment) and/or baseline risk of progression of AKI (creatinine increased over the previous 24 h by at least 20% to over 120 μmol/L) [13].In both trials [7,12], there were groups of patients with and without AKI.To ascertain the mortality count and total sample size for the no/not known AKI subgroup, the mortality count and total sample size of the AKI subgroup were subtracted from the overall mortality count and total sample size, respectively.
Post hoc, since we included studies combining higher protein and early physical rehabilitation, we added the subgroup analysis of studies with and without early physical rehabilitation.One study randomized patient to 3 groups (Group 1: usual care, Group 2: low protein + cycle ergometry, Group 3: high protein + cycle ergometry) [17], and we included groups 1 and 3 in our meta-analysis.

Data analysis
Dichotomous outcomes were presented as risk ratio (RR), while continuous outcomes were presented as mean difference (MD) or standardized mean difference (SMD).For AKI subgroup analysis on mortality outcome, we performed an additional analysis to present the effect measure as risk difference (RD) in order to obtain the number needed to harm (1/RD).The DerSimonian-Laird random-effect model was used to account for the different patients' characteristics, dosing, duration, and starting time of the protein delivery.Heterogeneity was quantified by the I 2 measure.Publication bias was visualized by the funnel plot.Egger's test was conducted for meta-analyses that included > 10 studies using STATA 16.1 (StataCorp LLC, Texas) [14].All meta-analyses and tests for subgroup differences were conducted using Rev-Man 5.4 (Cochrane IMS, Oxford, UK).A two-sided p value of < 0.05 was considered statistically significant, and a p value of < 0.10 was considered a trend.[15]

Trial sequential analysis
To control for type-I and type-II errors, TSA was performed using the TSA software (0.9.5.10 Beta, The Copenhagen Trial Unit, Denmark) with pre-specified parameters detailed in Additional file 1: Supplementary Methods.

Certainty of the evidence
The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) system was used to rate the certainty of evidence for outcomes analysed with TSA [16].The quality of the evidence was rated as high, moderate, low, and very low by considering the risk of bias, inconsistency, indirectness, imprecision, and publication bias.The percentage of diversityadjusted required information size (DARIS) achieved, and the TSA-adjusted 95% confidence interval for relative risk and mean difference were used to aid the assessment of imprecision in GRADE.GRADEpro was used to prepare the GRADE evidence profile table.

Study selection
Our search identified an additional 853 articles (391 from MEDLINE, 350 from EMBASE, and 113 from CENTRAL).After removing duplicates and article screening and review, we included 23 RCTs (an additional 4 RCTs [7,[17][18][19] from our previous SRMA).The detailed study selection flow is presented in Additional file 1: Fig. S1.The list of excluded studies and reasons for exclusion are presented in Additional file 1: Table S4.Our search on ClinicalTrials.govand personal files identified 13 ongoing or unpublished related trials (Additional file 1: Table S2).

Studies and patients' characteristics
Twenty-three RCTs with 3,303 patients were included.The study characteristics are summarized in Table 1.Sample sizes ranged from 20 to 1,301.Patients' baseline characteristics and the detailed nutritional data are summarized in Additional File 1: Tables S5 and S6.

Protein and energy delivery
Of the 23 included studies, 9 and 10 studies did not report the protein and energy delivered in g/kg BW/d or kcal/ kg BW/d, respectively.The pooled mean protein delivery for the higher vs lower protein group was 1.49 ± 0.48 vs 0.92 ± 0.30 g/kg BW/d (14 studies, n = 2439), respectively, resulting in a daily MD of 0.49 g/kg BW/d (95% confidence interval [CI] 0.37-0.61,p < 0.00001; I 2 = 94%) more protein delivery in the higher protein group.In contrast, the pooled mean energy delivery for the higher vs lower protein group was 17.48 ± 6.85 vs 16.60 ± 6.63 kcal/kg BW/d (13 studies, n = 2258), with no difference in daily energy delivery between groups (MD 0.13 kcal/kg BW/d, 95% CI − 1.25 to 1.52, p = 0.85;

Early physical rehabilitation delivery
Two studies combined high protein and early physical rehabilitation [17,18], and one study combined high protein and neuromuscular electrical muscle stimulation (NMES) [32], which are collectively named as combined early physical rehabilitation intervention.The details of the intervention are summarized in Additional file 1: Table S7.The NMES intervention was delivered in two 30-min sessions per day for up to 14 days.[32] For cycle ergometry, one study started immediately after randomization and delivered the intervention in two 15-min sessions/day for up to 21 days.[18] Another study started cycle ergometry within 24 h of randomization and delivered the intervention for up to 28 days, either passive cycling for 20 min/day or two 10-min sessions/day if a patient was able to cycle actively.[17]

Study quality assessments
The median CCN methodological quality score of included studies was 8 (out of 14 [higher score indicates higher quality]).A total of 10 studies had a methodological quality score of > 8 [7, 17, 21-24, 27, 29, 32, 36] (Additional file 1: Table S8).The ROB2 plots are presented in Additional file 1: Figure S3.In 21 studies that reported mortality outcomes, 4/21 (19%) studies were at low risk of bias, 14/21 (67%) had some concerns, and 3/21 (14.3%) were at high risk of bias.The biases mainly arose from the randomization process and selection of the reported results.

Results of the clinical outcomes
All outcomes are summarized in Additional file 1: Table S9 and Table S10.

Results of muscle mass and strength, discharge to rehabilitation facilities, self-reported quality of life physical function outcomes, and incidence of diarrhoea
No new studies were added to the meta-analysis on change in muscle mass, discharge location, and incidence of diarrhoea (Fig. 2a, 2c and 2e); therefore, findings are identical to our previous published meta-analysis [6].Notably, higher protein delivery is associated with a muscle loss attenuation (MD − 3.44% per week, 95% CI − 4.99 to − 1.90, p < 0.0001, I 2 = 16%; 5 studies; Fig. 2a).

Other subgroup analyses
No subgroup differences were detected between studies with low risk of bias and other risk of bias (Additional file 1: Fig. S7a-S7j) and studies that primarily used EN versus exclusive PN to increase protein delivery (data not shown).No subgroup differences were detected between single and multicentre studies (Additional file 1: Fig. S8a-8j).

Trial sequential analysis
Results of TSA are summarized in Table 2 and presented in Fig. 3 and Additional file 1: Figure S9, showing that the current systematic review did not achieve the required information sizes to detect the pre-specified effect sizes for overall mortality, infectious complications, ICU and hospital length of stay, change in muscle mass, handgrip strength, incidence of diarrhoea, and discharge to rehabilitation facilities, indicating that more trials are required for a definitive conclusion for these outcomes.
In patients with AKI, TSA confirmed the increase in mortality with high certainty.TSA revealed that further trials would be futile to detect a one-day difference in the duration of mechanical ventilation.

GRADE certainty assessments
Higher protein delivery did not affect overall mortality in critically ill patients (low certainty of evidence).On the contrary, higher protein delivery increased mortality among patients with AKI (high certainty of evidence).The certainty of evidence of the effect of higher protein on other outcomes is low to very low (Table 3).

Discussion
This updated SRMA with overall 23 RCTs (3303 patients) of higher versus lower protein delivery, mostly commenced within 3 days of ICU admission, and with similar energy delivery between groups, highlighted that higher protein delivery was not associated with improvements in clinical outcomes (overall mortality, infectious complications, ICU, and hospital length of stays) as well as muscle strength, discharge location, and incidence of diarrhoea; however, TSA indicated that more trials are needed to further confirm these findings.Importantly, higher protein delivery was associated with increased mortality among patients with AKI, a result confirmed  The parallel lines in green are the boundaries of conventional meta-analysis (alpha 5%), and the boundaries of benefit and harm are boundaries of conventional meta-analysis adjusted for between-trial heterogeneity and multiple statistical testing (TSA boundaries).A treatment effect outside the TSA boundaries of benefit/harm indicates reliable evidence for a treatment effect, and a treatment effect within the futility zone (the triangle between the parallel lines) indicates that there is reliable evidence of no treatment effect.DARIS: diversity adjusted required information size is the calculated optimum sample size for statistical inference, MID: minimally important difference, RRR: relative risk reduction, TSA: trial sequential analysis Trial sequential analysis revealed that only 50.8% of the required information sizes were attained to detect a 5-kg difference in handgrip strength r Sample size is too small for trial sequential analysis to determine the required information size to detect a relative risk reduction of 10% s Funnel plot asymmetry, Egger's test p = 0.0308 (Additional file 1: Figure S9c) by TSA.A non-statistically significant trend towards reduced mortality was found in subgroup of patients with no/not known AKI, and further trials in non-AKI are warranted to confirm this finding.This SRMA also found that higher protein delivery may attenuate muscle loss by about 3.4% per week; however, this finding was reported in a small number of studies, and TSA demonstrated a type-1 error, indicating that more studies are needed to improve the certainty of this finding.Furthermore, the combination of high protein delivery and early physical rehabilitation may improve self-reported quality of life physical function measures at day 90 after ICU admission (2 studies).Higher protein also significantly increased serum urea, urinary urea nitrogen, and lymphocyte count.

Interpretation of the results in the context of other evidence
Our findings suggest that higher protein delivery may harm patients with AKI.Despite the heterogeneous definition of AKI in the three meta-analysed studies, the direction of the results is similar (I 2 = 0%), particularly from the two included multicentre RCTs [7,13].In this context, using isotope technique, Chapple et al. recently revealed that critically ill patients exhibited a markedly blunted muscle protein synthesis or anabolic resistance compared to healthy controls [38].Notably, the incorporation of amino acids into the myofibrillar protein was 60% lower compared to a healthy control group.The reduced capacity to utilize protein during the acute phase of critical illness observed by Chapple et al., together with our findings of significantly higher serum and urinary urea as a result of higher protein provision, leads to a hypothesis that surplus protein may not be used for anabolism but is converted to urea for excretion.Higher urea levels may increase the metabolic burden of critically ill patients, particularly those with AKI, which may be one of the contributing factors to increased mortality in AKI patients, as demonstrated in our meta-analysis.Although a statistically significant mean increase of 2.31 mmol/L of serum urea or a mean increase of 5.55 g of urinary urea nitrogen may not be clinically significant in general, its clinical significance in critically ill patients with AKI remains unknown.Hence, the current findings have significant clinical implications, especially when considering the fact that current guidelines recommend higher protein delivery for critically ill patients with AKI, which should be carefully revised [39][40][41].In contrast, the finding of non-statistically trend towards lowered mortality of higher protein delivery in patients with no/ not known AKI requires further investigations.
Recent observational studies with robust statistical adjustments have examined protein delivery to critically ill patients during their first 5-7 days in the ICU.They have found that providing higher levels of protein, as opposed to medium or standard levels, does not lead to improved clinical outcomes and may even be harmful.One study by Hartl et al. involving 16,489 patients showed that protein delivery of 0.8-1.2g/kg BW/d after 5 days of ICU admission resulted in lower hospital mortality compared to exclusively low protein intake (< 0.8 g/ kg BW/d for ≤ 11 days).However, there was no further improvement in mortality when compared to early high protein intake (> 1.2 g/kg from day 1) [42].Similarly, Matejovic et al. studied 1,172 patients with ≥ 5 days ICU-LOS and found that moderate nutrition dose (10-20 kcal/kg for energy and 0.8-1.2g/kg for protein) improved patient weaning and reduced 90-day mortality compared to exclusively low nutrition intake (< 10 kcal/ kg BW + < 0.8 g/kg BW/d).Yet, there was no additional benefit when comparing moderate to high nutrition dose (> 20 kcal/kg BW/day + > 1.2 g/kg BW/d) [43].Lastly, Lin et al. studied 2,191 patients with ≥ 7 ICU-LOS and found that both high (1.68 g/kg BW/d) and low (0.38 g/ kg BW/d) protein intake, compared to medium protein intake (0.8 g/kg BW/d), were associated with increased 28-day mortality [44].Overall, these findings align with the conclusion that higher protein intake (around 1.5 g/ kg BW/d) during the first week of critical illness does not offer additional benefits in improving clinical outcomes for critically ill patients.
While no significant differences in clinical outcomes were observed, higher protein delivery may help attenuate muscle loss.Combined with early physical rehabilitation, it could potentially improve long-term self-reported quality of life physical function score.In this context, a recent systematic review among healthy and non-critically ill patients found that higher protein was associated with increased lean body mass; however, the rate of lean body mass gain plateaued beyond 1.3 g/kg BW/day without resistance training [45].It is plausible that certain subgroups of critically ill patients, particularly those who receive early physical rehabilitation, may experience greater muscle loss attenuation, ultimately enhancing their physical function.Similar findings were evident in ICU patients with traumatic brain injury, where those with greater quadriceps muscle thickness reported better physical function.[46] Another study linked greater lean mass with improved gait speed and 6-min walk distance in survivors of acute respiratory distress syndrome [47].However, these objective outcomes were not assessed in the studies included in our systematic review.Nevertheless, the observed muscle loss attenuation may be a type-1 error, as indicated by TSA, underscoring the need for more studies to validate this finding.Similarly, the improvement in self-reported physical function scores with combination therapy only trended towards significance and primarily originated from small studies.Ongoing trials with combinations of high protein and early physical rehabilitation (excluding patients with AKI and not on kidney replacement therapy), such as the NEXIS (NCT03021902; registered 16 Jan 2017) and EFFORT-X (NCT04261543; registered 7 Feb 2020), which assess physical function outcomes with objective measures such as the 6-min walk test and short physical performance battery test, will provide further insights into the impact of higher protein delivery on physical function outcomes in non-AKI patients.

Strength and limitations
The strength of our work lies in the comprehensive search and analysis and the predefined analysis plan for meta-analysis and TSA, all of which increase the transparency of information.In addition, excluding RCTs with different energy delivery between groups or pharmaconutrition interventions enabled us to focus solely on examining the effects of protein dosage.Furthermore, the use of TSA enabled us to detect the risk of type-1 or type-2 errors in our findings.The DARIS estimated from TSA will also inform the sample size needed for adequately powered future trials.Additionally, including extensive biochemical outcomes helped us elucidate the effects of higher protein delivery on metabolic parameters in critically ill patients.
Our work has several limitations.First, the included studies are heterogeneous in terms of the study population, dosage, timing, and routes of protein delivery.However, the included trials generally enrolled severely ill patients and primarily started intervention within 3 days of ICU admission.The subgroup analysis based on primarily EN vs exclusive PN/IV amino acids is consistent with the findings of the main analysis.The protein separation of approximately 0.49 g/kg BW/d with similar energy delivery between groups also ensures that the effect of protein was studied.Second, the three studies included in our analysis use varying definitions of AKI, which could limit the applicability of our findings in clinical practice.However, all the definitions identified AKI through an acute rise in serum creatinine levels.We recommend using the KDIGO definition of AKI [48] to guide protein delivery, as recent evidence showed that higher protein delivery is associated with increased mortality across all AKI stages, especially in patients who did not receive kidney replacement therapy [49].Third, the number of studies with combinations of high protein and early physical rehabilitation intervention was limited, and the result is mainly attributed to one single-centre study with a high risk of bias [18].Lastly, the certainty of evidence for most outcomes was assessed as low to very low due to the risk of bias and imprecision.Hence, more high-quality studies are warranted, especially studies with combined interventions (high protein and early physical rehabilitation).

Conclusion
The present updated SRMA demonstrated that a higher protein delivery in the acute phase of critical illness has no effects on relevant clinical outcomes but significantly increased urea levels.Importantly, higher protein delivery increased mortality rates among AKI patients with high certainty, while its effect among non-AKI patients requires further investigation.In contrast, higher protein delivery may attenuate the loss of muscle mass, and the combination of high protein delivery and early physical rehabilitation may further improve self-reported physical function; however, these effects were only reported in a small number of studies of moderate to low quality.Future trials that combine high protein with early physical rehabilitation (in non-AKI patients) and assess objective physical function outcomes are warranted.Meanwhile, protein delivery should be carefully monitored in critically ill patients with AKI.

Fig. 1
Fig. 1 Meta-analysis of clinical outcomes.a Overall mortality (all patients), b Overall mortality (subgroup analysis of no/not known AKI vs AKI)*, c infectious complications (no change from previous meta-analysis), d ICU length of stay, e hospital length of stay, f duration of mechanical ventilation.AKI: acute kidney injury.*Note: b, c: AKI subgroup: mortality from Doig 2015 is 90-d mortality from their secondary publication [13].Definitions: Singer 2007: AKI-50% decrease in GFR, a doubling of serum creatinine or an increase of creatinine to 3.5 mg/dL (309.4 umol/L); Doig 2015 (mortality of patients with kidney dysfunction or risk of progression of AKI from Doig 2015 is 90-d mortality from their secondary publication [13]): Baseline kidney dysfunction-creatinine at time of enrolment > 168 μmol/L (by Gordon Bernard's "Brussels Table"), Risk of progression of AKI at enrolment-a rise in creatinine over the previous 24 h by at least 20% to over 120 μmol/L; Heyland 2023: AKI-patients who met the criteria of KDIGO: stage 1 is at least 26•52 μmol/L increase in serum creatinine from baseline within 48 h or 1•5-1•9 times baseline within 7 days, stage 2 is 2•0-2•9 times baseline within 7 days, or stage 3 is three times or more baseline within 7 days or increase to at least 353•6 μmol/L with an acute increase of more than 44•2 μmol/L.c: To ascertain the mortality count and total sample size for the no/not known AKI subgroup for Doig 2015 and Heyland 2023, the mortality count and total sample size of the AKI subgroup were subtracted from the overall mortality count and total sample size, respectively; mortality for Doig 2015 is 90-day mortality

(Fig. 2
See figure on next page.)Meta-analysis of other outcomes.a Percentage of muscle change per week (no changes from previous meta-analysis), b handgrip strength, c discharge to rehabilitation facilities (no changes from previous meta-analysis), d self-reported quality of life physical function at day 90, e incidence of diarrhoea (no changes from previous meta-analysis).Note: b Fetterplace 2018: the best handgrip strength at awakening, ICU discharge, or day 15, Ferrie 2015: handgrip strength at day 7.Unable to analyse handgrip strength from Azevedo 2019 because unknown sample size for male and female.d The quality of life (QOL) outcomes reported by the studies were: Doig 2015: RAND-36 general health and physical function at day 90; Azevedo 2019: SF-36 physical component summary (PCS) score at 3 and 6 month; Badjatia 2010: fatigue, lower extremity mobility, and cognition outcomes based on the Neuro-QoL questionnaires administered on post-bleed day 90; Chapple 2020: EQ-5D-5L score for mobility, self-care, usual activities, pain/discomfort, anxiety/depression, and the result of the EQ-5D-5L visual analogue scale, all at day 90, Azevedo 2021: SF-36 physical component score at day 3 and 6 month (see Additional file 1: Table

D 2 :Fig. 3
Fig. 3 Trial Sequential Analysis of Clinical Outcomes.a Overall mortality in all patients (21 studies, n = 3125), b overall mortality in patients with acute kidney injury before protein intervention (3 studies, n = 428), c infectious complications (7 studies, n = 642), d intensive care unit length of stay (16 studies, n = 2516), e hospital length of stay (11 studies, n = 2130), f duration of mechanical ventilation (13 studies, n = 2360).TSA was analysed using DerSimonian and Laird random-effects model.The Z curve in blue measures the treatment effect (pooled relative risk).The parallel lines in green are the boundaries of conventional meta-analysis (alpha 5%), and the boundaries of benefit and harm are boundaries of conventional meta-analysis adjusted for between-trial heterogeneity and multiple statistical testing (TSA boundaries).A treatment effect outside the TSA boundaries of benefit/harm indicates reliable evidence for a treatment effect, and a treatment effect within the futility zone (the triangle between the parallel lines) indicates that there is reliable evidence of no treatment effect.DARIS: diversity adjusted required information size is the calculated optimum sample size for statistical inference, MID: minimally important difference, RRR: relative risk reduction, TSA: trial sequential analysis

Table 1
Study population, nutrition route, and timing of intervention

Table 1 (
continued) AA: amino acid, APACHE II: Acute Physiology and Chronic Health Evaluation II, BMI: body mass index, d: day(s), DC: discharge, EN: enteral nutrition, GCS: Glasgow Coma Scale, h: hour, IC: indirect calorimetry, ICU: intensive care unit, IV: intravenous, LOS: length of stay, Mixed: medical and surgical populations, MV: mechanical ventilation, PN: parenteral nutrition, SPN: supplemental parenteral nutrition *N: number of patients analysed (total number of patients in 23 included studies # 'High' nutrition risk is defined by one of the following risk factors: BMI

Table 2
Summary of results of trial sequential analyses

Table 3
GRADE certainty assessment and summary of findings table

Risk of bias Inconsistency Indirectness Imprecision Publication bias Overall certainty of evidence Study event rates (%) Relative effect (95% CI) Anticipated absolute effects With placebo With mortality
had low risk of bias, the moderate study had some concerns, the smallest study had high risk of bias.Overall, the results are consistent and therefore we rated this as not serious Trial sequential analysis revealed that only 8.6% of the required information sizes were attained to detect a relative risk reduction of 10% b Trial sequential analysis revealed that only 25.7% of the required information sizes were attained to detect a relative risk reduction of 10% c The largest study d Trial sequential analysis confirmed the increase in mortality with high certainty e All studies had some concerns f